Detecting ‘Heat’ with Light: Luminescent Sensors for Capsaicin

1. Detecting ‘Heat’ with Light: Luminescent Sensors for Capsaicin Christopher G. Gulgas

2. Chemistry review: Hydrogen bonding • Molecules with O-H bonds or N-H bonds can “hydrogen bond” to other molecules δ- δ+ δ+

3. Chemistry review: Hydrogen bonding δ- δ-

4. Chemistry review: Lipids • Lipids are hydrophobic molecules usually containing long carbon chains mostly saturated with hydrogen • They are nonpolar molecules and would disrupt the hydrogen-bonding network in bulk water

5. Part 1 - Capsaicin “oily” alkyl chain (not rigid) = area of (+) charge = area of (-) charge

6. Capsaicin • Member of the vanilloids • Biological properties1-2 • Anticancer • Antioxidants • Analgesic (pain relief creams) • Anti-inflammatory • Antimicrobial • Promote energy consumption and decrease accumulation of fats Barbero, G.F. et al. J. Agric. Food Chem. 2010, 58, 3342-3349. Rusterholz, D.B. J. Chem. Educ. 2006, 83, 1809-1815.

7. Naturally-occurring capsaicinoids • Capsaicin • Dihydrocapsaicin • Nordihydrocapsaicin • Homocapsaicin • Homodihydrocapsaicin

8. What’s the effect of shortening the “oily” chain?

9. Capsaicin3 • Nelson evaluated pungency • Relative pungency determined by the noting the minimum amount of capsaicinoid resulting in “a distinct burning on the tip of the tongue”. • 0.00013 mg (130 ng) of capsaicin could be detected, and provided the standard for comparison • Alcoholic solutions were made of the capsaicinoids, and concentrations were varied until the same degree of burning sensation was detected from one drop. • Individual descriptions • n=0 (not pungent) • n=1 (very slightly pungent) • n=2 (somewhat pungent) • n=5 (far more pungent than earlier members) • n=6 (very pungent) • n=7 (violently pungent) “about equal to capsaicin, but its property of causing sneezing and coughing is probably not quite so great” • n=8 (extremely pungent – more disagreeable to handle than n=7) • n=9 (extremely pungent; coughing and sneezing) • n=10 (different pungency, not so immediately apparent, effecting the back of the tongue and throat) Comparative Pungencies n=5 5 n=6 25 n=7 75 n=8 100 n=9 50 n=10 25 n=11 25 3. Nelson, E.K. et al. J. Am. Chem. Soc. 1919, 41, 2121-2130.

10. Hydrophobic effect = “fat/oily” region

11. Capsaicinoids as Pain Relievers2,4 • Capsaicin binds to the TRPV1 receptor protein (transient receptor potential vanilloid), an ion channel protein4 • TRPV1 is located in sensory nerve fibers throughout the body, activated by vanilloids or heat (> 42°C) • Activation leads to an opening of the ion channel, and calcium appears to be the primary ion involved • Acidic conditions enhance the effect • Desensitization to the effects of capsaicin have been observed (with or without permanent damage to the nerve cells) • Structural analogs to capsaicin have been synthesized, and their relative potency in binding to the TRPV1 receptor studied • The goal is to design highly potent, less pungent desensitizing drugs to reduce pain 4. Caterina, M.J. et al. Nature 1997, 389, 816-824.

12. Capsaicinoids as Pain Relievers2 • Replacement of the amide functionality with a stronger hydrogen-bond donating group has resulted in higher relative potency (RP)

13. Capsaicinoid synthesis

14. Current Research Goal • Capsaicinoids are effective for pain relief, and have favorable biological properties • Capsaicinoid concentrations can be measured readily by HPLC methods • Questions • Can we design a luminescent sensor molecule for the detection of capsaicinoids in solution? • Will undergraduate researchers learn structure-activity relationships relating to drug discovery and design by synthesizing and evaluating these sensor molecules? • Goal • I want to have a molecule (a metal complex) that detects the capsaicinoid family through luminescence. Nothing exists like this for capsaicinoids. • The creation of a suitable metal complex will open up further research in biological systems, potentially

15. Quantification of Capsaicin - HPLC • High Pressure Liquid Chromatography • Mixtures are separated and components are quantified • Separation occurs through molecular properties such as polarity or size • Molecules interact with the column and the solvent • Can be expensive and time consuming, but is the industry standard

16. 5 x 10 2 1.5 Absorbance at 270 nm (mAU) 1 0.5 0 2 3 4 5 6 7 8 9 Retention Time (min) Example of data Doxylamine Dextromethorphan Acetaminophen Guaifenesin Caffeine Aspirin **Courtesy of Dr. Sarah Porter

17. Part 2 – Lanthanide-based Sensor • A = analyte open coordination sites A

18. EDTA • Ethylenediaminetetraacetic acid • Usually incorporated in foods as “disodium EDTA” • Sequesters metal ions in paints/dyes, in foods, and in the body

19. Lanthanide properties • Lanthanide (III) ions are hard acids, preferring oxygen donors, and stable complexes are formed in aqueous solution only with multidentateligands. • Lanthanide ions typically coordinate 8-9 donor atoms • Many lanthanides are luminescent in the visible range and have sharp emission lines http://perso.univ-rennes1.fr/martinus.werts/lanthanides/ln_shine.html

20. Lanthanide luminescence • Direct excitation is not efficient; a light-harvesting antenna is typically used for augmented luminescence • Millisecond lifetimes allow for time resolved fluorescence measurements • Ln3+-antenna distance is important Energy Transfer hv2 Antenna Spacer Ln3+ hv • De-excitation of the excited lanthanide can occur through non-emissive pathway (OH, NH oscillators) Petoud, S.; Cohen, S. M.; Bünzli, J.-C. G.; Raymond, K. N. J. Am. Chem. Soc.2003, 125, 13324-13325.

21. Chelate Synthesis 92% 42%

22. Binding capsaicinoids How do we begin to understand what is going on? Luminescence experiments will shed some light on the subject.

23. Luminescence Experiments detector • Emission Scan • One wavelength of excitation • Scan a range of wavelengths for emission intensity of the solution • Excitation Scan • One wavelength of emission measured • Use a range of wavelengths for excitation of the solution sample excitation source detector sample excitation source

24. Proof-of-concept with cap1 • Over 500x luminescence augmentation!! [Tb(1)]+ = 5.0 x 10-5 M

26. Excitation spectrum (545 emission) [Tb(1)]+ = 5.0 x 10-5 M

27. Compare 290 nm exc. w/ 315 nm exc. over the cap1 concentration range

28. Cap1 w/ [Eu(1)]+ • Decrease in luminescence upon excitation at 270 nm, but corresponding increases at 317 nm and 365 nm • Notice maximum increase at 317 nm occurs at 20 eq (compare to [Tb(1)]+ [Eu(1)]+ = 5.0 x 10-5 M

30. Conclusion of initial experiments • The vanilloid structure is capable of sensitizing lanthanide emissions • Simple capsaicinoids can be detected by EDTA-based lanthanide chelates through easily observed luminescence signaling • Ratiometric fluorescence detection is plausible • Methods are in place for characterizing the luminescent response and nature of the complex(es) formed in solution • There remain some questions: How does cap1 bind to the metal complex?

31. How does cap1 bind to [Ln(1)]+ ? • Does cap1 bind through its two oxygen atoms and displace water molecules?

32. How does cap1 bind to [Ln(1)]+ ? H2O D2O • Luminescence lifetime titration • Compare rates of luminescence decay • A larger discrepancy between H2O and D2O means more water molecules are coordinated Small # of coordinated water molecules Large # of coordinated water molecules

33. How does cap1 bind to [Ln(1)]+ ? • Luminescence lifetime titration • Compare rates of luminescence decay • A larger discrepancy between H2O and D2O means more water molecules are coordinated

34. How does cap1 bind to [Ln(1)]+ ? • Cap1 does not appear to displace more than one water molecule, and only in the case of Eu So exactly how does cap1 bind?

35. How does cap1 bind to [Ln(1)]+ ? • Does cap1 bind the lanthanide through its two oxygens in a bidentate fashion? We would expect a shift in the absorbance wavelength of cap1 if this binding mode occurs!

36. How does cap1 bind to [Ln(1)]+ ? • Must bind through some alternative way!

37. How does cap1 bind to [Ln(1)]+ ? • How many molecules of cap1 can bind to one complex of [Ln(1)]+ ? (Job’s plot data)

38. How does cap1 bind to [Ln(1)]+ ? • How can we figure this out? • Work with some other similar lanthanide complexes and capsaicinoids • Obtain crystal structures of [Ln(1)]+ with cap1

39. Cap1 binding to naked Ln3+ ions • Luminescence enhancement observed on the order of that observed in our chelates • Evidence for partial removal of coordinated water molecules

40. Designing new lanthanide complexes • We want to be able to bind and detect the naturally-occurring capsaicinoids (all have a long alkyl chain)

41. Synthetic pathway [Ln(3)]+ [Ln(2)]+

42. 2 more complexes • Structural rigidity of aromatic ring system eases purification • [Ln(4)]+ is a precursor for expansion

43. Luminescence studies with [Tb(2)]+ • Luminescence augmentation is not as dramatic • Ligand 2 is an antenna (see intensity at 0 eq)

44. Luminescence studies with [Tb(2)]+ • Excitation at 315 nm grows in (similar to [Tb(1)]+

45. Future Work • Figure out what the heck is going on (really) • Chelate design must evolve! • we currently focus on binding only one region of the capsaicin structure • Incorporate functional groups on the chelate to hydrogen bond to the amide of capsaicinoids, and utilize the hydrophobic effect • Synthesize and study other capsaicinoids and compare binding constants/stoichiometry

46. Ultimate design • Subject to change as we learn more!

47. Acknowledgement Longwood University - Department of Chemistry and Physics (research funding and start-up) - Cook-Cole fund, awarded January 2011